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Penetrating atherosclerotic ulcer (PAU) is defined as ulceration of an aortic atherosclerotic plaque penetrating through the internal elastic lamina into the media.1–3 PAU accounts for 2% to 7% of all acute aortic syndrome.4 PAU is at risk of intramural hematoma, pseudoaneurysm, aortic rupture, or an acute aortic dissection.5 Symptomatic PAU has to be assumed to indicate an emergency because the adventitia is reached and aortic rupture is expected.5 The rupture rate of symptomatic PAU has been reported to be as high as …

A 35-year-old Japanese woman for whom a previous health checkup showed normal blood pressure and urinalysis results without serological abnormalities developed nephrotic syndrome with severe hypertension at 15 gestational weeks. The renal biopsy performed at 17 weeks of gestation showed severe glomerular capillary endotheliosis. By means of electron microscopy, no electron-dense deposits were observed in glomeruli, and foot-process arrangement was normal. Histological findings indicated the patient's glomerular damage was caused by the mechanisms of preeclampsia. The patient underwent an elective abortion at 18 weeks of gestation. Clinical abnormalities vanished completely within 3 months after the elective abortion, which provided additional evidence that proteinuria and hypertension were caused purely by pregnancy. In general, the term preeclampsia refers to new onset of hypertension and proteinuria after 20 weeks of gestation. When proteinuria or hypertension is newly observed before 20 weeks of gestation, they are practically associated with triploidy, trophoblastic disease, or antiphospholipid syndrome. However, our case was not associated with them. Therefore, we called this case "pure" preeclampsia. We confirm the notion for the first time that preeclampsia associated with glomerular capillary endotheliosis can occur before 20 weeks of gestation. In addition, this report describes the earliest onset of preeclampsia compared with previously published reports. We also discuss causes of preeclampsia in early gestation and refer to the issue of the application of renal biopsies during pregnancy.

Systemic aldosterone plays an important role in the development of the microvascular disease and glomerular damage of the kidney in patients with diabetes mellitus and hyperlipidemia. Here, we investigated the possibility of local production of aldosterone in the kidney, using human primary glomerular mesangial cells. These cells produced both pregnenolone and aldosterone measured by specific radioimmunoassay and/or gas chromatography/mass spectrometry (GC/MS) methods. The production of both steroids was significantly stimulated by treatment with LDL, while angiotensin II had a synergistic effect. Adrenocorticotropic hormone (ACTH) and (Bu)2cAMP, on the other hand, failed to stimulate aldosterone production by these cells, suggesting that the local production of this steroid by mesangial cells is regulated differently from that of adrenal zona glomerulosa cells. Mesangial cells expressed the mRNA of the LDL receptor and steroidogenic enzymes, such as P450scc, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 21-hydroxylase and CYP11B2. Mesangial cells also expressed mRNA of the mineralocorticoid receptor (MR), and LDL stimulated its abundance by three-fold, while spironolactone, a completive antagonist of aldosterone, completely abolished this LDL effect. Since MR is a known mineralocorticoid-responsive gene as well as an intracellular receptor molecule for this steroid, these results suggest that locally produced aldosterone is biologically active, stimulating the transcription rates of the mineralocorticoid-responsive genes by activating the MR in mesangial cells. These pieces of evidence indicate that human mesangial cells are an aldosterone-producing tissue in which LDL plays a major regulatory role. Therefore, human renal mesangial endocrine system may contribute to local aldosterone concentrations and effects in the renal glomerulus independently of the systemic renin--angiotensin--aldosterone system and may participate in the development and progression of glomerular damage in several pathologic conditions.

Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511GA (G171S), 556CT (R186X; X represents the translational stop codon), 556CG (R186G), 740AG (K247R), and 1149GT (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6alpha-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6alpha-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6alpha-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556CT (R186X) and 556CG (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.

Metabolic capacities for debrisoquin, sparteine, mephenytoin, nifedipine, and midazolam, which are substrates of polymorphic CYP2D6, CYP2C19, and CYP3A, have been reported to exhibit, in many cases, remarkable interindividual and ethnic differences. These ethnic differences are partly associated with genetic differences. In the case of the drug transporter ABCB1/MDR1, interindividual differences in its transporter activities toward various clinical drugs are also attributed to several ABCB1/MDR1 genetic polymorphisms. In this review, the existence and frequency of various low-activity alleles of drug metabolizing enzymes as well as populational drug metabolic capacities are compared among several different races or ethnicities. Distribution of nonsynonymous ABCB1/MDR1 SNPs and haplotype frequency in various races are summarized, with the association of nonsynonymous SNPs with large functional alterations as a rare event.

Amiodarone is a potent Class III antiarrhythmic drug. The N-deethylation of amiodarone to desethylamiodarone is known to be catalyzed by cytochrome P450 (CYP) 2C8. In the present study, amiodarone N-deethylation by the CYP2C8s, CYP2C8*1 (wild-type), CYP2C8*3, and CYP2C8 P404A (Pro404Ala substitution in exon 8), was investigated by their transient expression in Hep G2 cells. The expression levels of CYP2C8*1 and CYP2C8*3 were similar, whereas the level of CYP2C8 P404A was 55.6% of that of CYP2C8*1. The kinetic parameters of amiodarone N-deethylation were obtained by means of Lineweaver-Burk analysis. The intrinsic clearance (Vmax/Km, per mg of microsomal protein) of amiodarone by CYP2C8 P404A but not CYP2C8*3 was significantly (48.7%) less than that of CYP2C8*1. These results suggest that CYP2C8 P404A but not CYP2C8*3 is less effective in the N-deethylation of amiodarone.

Summary: Novel single nucleotide polymorphisms (SNPs) were found in introns 1, 3, 4, 5, and 7 of the gene encoding cytochrome (CYP) 2D6 in 90 Japanese subjects. Some of these SNPs were associated with C2850T and/or C100T, which are key SNPs for CYP2D6*2 and CYP2D6*10, respectively. The SNPs in the CYP2D6 gene (ACCESSION NUMBER, M33388) were designated as follows: SNP, MPJ6_2D6010 (IVS1-138C > G) associated with C2850T; SNP, MPJ6_2D6012 (IVS1-41 T > G) associated with C100T, C2850T and G4180C; SNP, MPJ6_2D6016 (IVS3 + 32A > G); SNP, MPJ6_2D6017 (IVS4-17C > G); SNP, MPJ6_2D6018 (IVS4-17C > T); SNP, MPJ6_2D6020 (IVS5 + 46G > A); SNP, MPJ6_2D6021 (IVS5-49 T > C); SNP, MPJ6_2D6023 (IVS7 + 40A > C) associated with both C100T, C2850T and G4180C; SNP, MPJ6_2D6024 (IVS7 + 64 T > C); and SNP, MPJ6_2D6025 (IVS7-9C > T) associated with C2850T. These SNPs are convenient for allele typing and should be further investigated for their potential effects on transcription, such as splicing.

RA175, a new member of the immunoglobulin superfamily, is highly expressed during neuronal differentiation of P19 embryonal carcinoma cells. In situ hybridization showed that RA175 mRNA was detected in the developing nervous system, as well as the epithelium of the various non-neuronal tissues of mouse embryo. In contrast with the epithelia of the non-neuronal tissues, RA175 mRNA was not co-expressed with Sonic hedgehog mRNA and Patched mRNA during brain morphogenesis. RA175 mRNA was highly expressed in the anterior horn cells and the peripheral nervous system at embryonic day (E) 11.5 and in the central nervous system at E14.5-E18.5, but its expression decreased after birth and was undetectable in the adult mouse brain.

By sequencing genomic DNA from 73 established cell lines derived from Japanese individuals, we detected 9 single nucleotide polymorphisms (SNPs) in the CYP2C8 gene. Of them, 3 exonic SNPs resulted in amino acid alterations (g416a, R139K; a1196g, K399R; c1210g, P404A). The first two alterations were detected concurrently in one cell line and thought to be the same as CYP2C8*3. To examine the effects of these amino acid alterations on CYP2C8 function, wild-type and four types of variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R and P404A) were transfected into Hep G2 cells and their paclitaxel 6a-hydroxylase activities were determined in vitro. Km values were not significantly different from that of the wild-type in any of the variants studied. The variant R139K/K399R showed reduced values for Vmax and clearance (Vmax/Km) similar to those of its single variant, R139K. The variant P404A also showed a significantly lowered clearance due to reduced level of protein expression. These results suggest that not only the double variant (R139K/K399R, CYP2C8*3) but also our novel variant P404A in the CYP2C8 gene are less efficient in paclitaxel metabolism.

The members of the cytochrome P450 (CYP) 3A subfamily play an important role in the metabolism of more than 50% of the drugs metabolized by CYPs. Among the CYP3A members, CYP3A5 is known to exhibit polymorphic expression within the human liver. We hypothesized that a single nucleotide polymorphism (SNP) in the 5'-regulatory region of the CYP3A5 gene might be the cause of CYP3A5 polymorphic expression. Due to the existence of “CYP3AP1, ” a highly homologous sequence to the CYP3A5 gene, it was necessary to make specific primers to the CYP3A5 gene. In the present study, we designed a series of oligonucleotide primers for sequencing the proximal promoter region of the CYP3A5 gene in order to search for the putative regulatory single nucleotide polymorphism. We examined 86 established cell lines derived from Japanese individuals as a representation of the Japanese population. However, no SNP was detected in the promoter region of the CYP3A5 gene isolated from the cell lines used, suggesting other causal factors for the observed polymorphism of CYP3A5-dependent drug metabolism.

We previously found that thiamine mitigates metabolic disorders in spontaneously hypertensive rats, harboring defects in glucose and fatty acid metabolism. Mutation of thiamine transporter gene SLC19A2 is linked to type 2 diabetes mellitus. The current study extends our hypothesis that thiamine intervention may impact metabolic abnormalities in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, exhibiting obesity and metabolic disorders similar to human metabolic syndrome. Male OLETF rats (4 wk old) were given free access to water containing either 0.2% or 0% of thiamine for 21 and 51 wk. At the end of treatment, blood parameters and cardiac functions were analyzed. After sacrifice, organs weights, histological findings, and hepatic pyruvate dehydrogenase (PDH) activity in the liver were evaluated. Thiamine intervention averted obesity and prevented metabolic disorders in OLETF rats which accompanied mitigation of reduced lipid oxidation and increased hepatic PDH activity. Histological evaluation revealed that thiamine alleviated adipocyte hypertrophy, steatosis in the liver, heart, and skeletal muscle, sinusoidal fibrosis with formation of basement membranes (called pseudocapillarization) which accompanied significantly reduced expression of laminin β1 and nidogen-1 mRNA, interstitial fibrosis in the heart and kidney, fatty degeneration in the pancreas, thickening of the basement membrane of the vasculature, and glomerulopathy and mononuclear cell infiltration in the kidney. Cardiac and renal functions were preserved in thiamine treatment. Thiamine has a potential to prevent obesity and metabolic disorders in OLETF rats.

Seasonal variations in blood pressure (BP) have often been attributed to meteorological factors, especially changes in outdoor temperature. We evaluated the direct association between meteorological factors and circadian BP variability. Twenty-four-hour ambulatory BP was monitored continuously for 7 days in 158 subjects. Mean awake, asleep, morning (first 2 h after waking) BP, prewaking morning BP surge (morning systolic BP (SBP)-mean SBP during the 2-h period before waking) and nocturnal BP decline were measured each day. We compared BP values for the lowest and highest days with regard to the daily mean outdoor temperature and mean atmospheric pressure. Morning BP and prewaking morning BP surge on the coldest day were significantly higher than those on the warmest day (morning SBP, 136.6 ± 1.6 vs. 133.1 ± 1.5 mm Hg, P = 0.002; morning diastolic BP, 84.4 ± 0.9 vs. 82.6 ± 0.9 mm Hg, P = 0.02; and prewaking morning BP surge, 20.8 ± 1.3 vs. 15.3 ± 1.3 mm Hg, P = 0.0004). The magnitude of nocturnal BP decline on the coldest day was significantly greater than that on the warmest day (15.8 ± 0.7 vs. 13.9 ± 0.7%, P = 0.01). Outdoor temperature is an important determinant of morning BP, prewaking morning BP surge and the magnitude of nocturnal BP decline. These findings may have important implications in management of hypertension and prevention of cardiovascular events.

During the course of sequencing for the CYP2D6 gene, we found a novel single nucleotide polymorphism of g.3318G>A (E383K) associated with CYP2D6*10, termed as CYP2D6*72. We also found a g.1611T>A (F120I) in the CYP2D6*49, which was previously identified as a CYP2D6*10-associated allele in an independent Japanese population. To clarify the effects of these novel CYP2D6*10 haplotypes on the functions of CYP2D6, kinetic analysis for dextromethorphan O-demethylation was performed using the Escherichia coli expression system and human liver microsomes. The V(max)/K(m) values for dextromethorphan O-demethylation catalyzed by recombinant CYP2D6 forms encoded by CYP2D6*10, CYP2D6*49, and CYP2D6*72 were 3.0, 0.5, and 1.3%, respectively, compared with that catalyzed by CYP2D6.1. Liver microsomes from a human subject genotyped as CYP2D6*10/*49 also showed a reduced dextromethorphan O-demethylase activity. CYP2D6.49 formed a 7-hydroxydextromethorphan, with a roughly similar V(max)/K(m) value to that of O-demethylation. These results suggest that these two CYP2D6*10 haplotypes are possible causes of interindividual variation in the activities and the substrate specificity of CYP2D6.

CYP2C8 is known to metabolize various drugs including an anticancer drug paclitaxel. Although large interindividual differences in CYP2C8 enzymatic activity and several nonsynonymous variations were reported, neither haplotype structures nor their associations with pharmacokinetic parameters of paclitaxel were reported.Haplotype structures of the CYP2C8 gene were inferred by an expectation-maximization based program using 40 genetic variations detected in 437 Japanese patients, which included cancer patients. Associations of the haplotypes and paclitaxel pharmacokinetic parameters were analyzed for 199 paclitaxel-administered cancer patients.Relatively strong linkage disequilibriums were observed throughout the CYP2C8 gene. We estimated 40 haplotypes without an amino-acid change and nine haplotypes with amino acid changes. The 40 haplotypes were classified into six groups based on network analysis. The patients with heterozygous *IG group haplotypes harboring several intronic variations showed a 2.5-fold higher median area under concentration-time curve of C3'-p-hydroxy-paclitaxel and a 1.6-fold higher median value of C3'-p-hydroxy-paclitaxel/paclitaxel area under concentration-time curve ratio than patients bearing no *IG group haplotypes (P0.001 for both comparisons by Mann-Whitney U-test). No statistically significant differences, however, were observed between patients with and without the *IG group (haplotypes) in clearance and area under concentration-time curve of paclitaxel, area under concentration-time curve of 6alpha-hydroxy-paclitaxel and 6alpha-, C3'-p-dihydroxy-paclitaxel, and area under concentration-time curve ratio of 6alpha-hydroxy-paclitaxel/paclitaxel.CYP2C8*IG group haplotypes were associated with increased area under concentration-time curve of C3'-p-hydroxy-paclitaxel and area under concentration-time curve ratio of C3'-p-hydroxy-paclitaxel/paclitaxel. Thus, *IG group haplotypes might be associated with reduced CYP2C8 activity, possibly through its reduced protein levels.

Although it is well recognized that acute mitral regurgitation (MR) is caused by acute myocardial infarction and infective endocarditis, blunt chest trauma is also, albeit rarely, reported as the cause. In most cases, acute MR provokes pulmonary edema and hemodynamic deterioration. We report a case of a 45-year-old man with atypical pulmonary edema caused by acute traumatic MR. The patient fell down the stairs and injured his …

Summary: Chimeric REP7/6 has been used as a marker of CYP2D6 deletion, such as for CYP2D6*5 . However, the CYP2D6*10D (* 10D ) haplotype found in a Japanese population consist of CYP2D6*10B , CYP2D7P -derived 3′-flanking region, and a chimeric repetitive sequence, CYP-REP7/6 (REP7/6) (Ishiguro et al. Clin. Chim. Acta. 2004: 347, 217–221). From our analysis, REP7/6 was found in 26 out of 254 Japanese subjects. Thus, the REP7/6-containing CYP2D6 genes ( 2D6 -REP7/6) were analyzed in detail. In order to specifically detect the 2D6 -REP7/6 structure, primers were designed in CYP2D6 intron 6 and the REP7/6 3′-flanking region. Among 26 subjects analyzed by PCR, 5 had 2D6 -REP7/6. The other 21 subjects were confirmed to have * 5 by another * 5 -specific primer set. Three out of five subjects with 2D6 -REP7/6 had the * 10D structure. However, further analysis by PCR and sequencing revealed that their haplotypes were further divided into tandem-type * 36 -* 10D (n = 2) and single-type * 10D (n = 1). The remaining two subjects had a novel type of a * 36 -containing defective structure that consists of CYP2D6*36 and 3′-flanking REP7/6 (single-type * 36 -REP7/6). Then, REP7/6 sequences in *5, *10D, *36-*10D , and single-type *36 were determined and classified into 5 types: types A to D for * 5 , type E for * 10D and * 36-*10D , and type F for * 36 . These findings could be useful for accurate determination of *5 and REP7/6-harboring aberrant CYP2D6 haplotypes.

The frequency of the CYP2D6*10 allele (100C>T) in the Japanese is relatively high (0.3-0.4), and the two *10-related genes, Ch1 (currently *10B) and Ch2 (*36), and their tandem arrangement Ch(2)-Ch(1) (*36-*10B) have been reported. Although the tandem form of *36-*10 is assumed to be a major form, no detailed information has been reported for its intervening and flanking regions. Thus in this study, the tandem-type *36-*10B and the single-type *10 were analyzed by long-range PCR and sequencing of the subsequent nested PCR products. The sequence of the entire *36-*10 region confirmed the recombination of CYP2D6*10 with CYP2D7P. Also, we found that most of the *10B-harboring haplotypes have the upstream *36 gene and that the majority of the remaining haplotypes are the single-type *10B. Haplotype frequencies of the single-type *10 and *36-*10B were 0.06 and 0.30, respectively, in the subjects analyzed. Additionally, several novel single nucleotide polymorphisms (SNPs) were found in the *36 region and several *36 haplotypes were identified. This sequence information is an important addition to the CYP2D6 sequence data that was obtained by the human genome project.

We report a 35-year-old woman with active acromegaly despite pituitary surgery and irradiation who received continuous octreotide LAR treatment for the control of GH excess until discovery of her pregnancy. The patient delivered a healthy boy following an uneventful pregnancy after discontinuing octreotide LAR as soon as possible at the early phase of pregnancy. Despite a substantial maternal-fetal transfer of octreotide, postnatal development was normal at 3 years of age. In almost all previously described cases, octreotide was discontinued after pregnancy was confirmed. No side-effects of mother or fetus have been reported. Octreotide treatment in pregnancy seems to be feasible and safe. Due to the still-limited number of reported cases treated with octreotide LAR, the potential benefits of octreotide LAR treatment should be weighed carefully against its possible risks.

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one-third of the medicines in current clinical use and exhibits genetic polymorphism with interindividual differences in metabolic activity. To precisely investigate the effect of CYP2D6*10B and CYP2D6*36 frequently found in Oriental populations on mexiletine metabolism in vitro, CYP2D6 proteins of wild-type (CYP2D6.1) and variants (CYP2D6.10 and CYP2D6.36) were heterologously expressed in yeast cells and their mexiletine p - and 2-methyl hydroxylation activities were determined. Both variant CYP2D6 enzymes showed a drastic reduction of CYP2D6 holo- and apoproteins compared with those of CYP2D6.1. Mexiletine p - and 2-methyl hydroxylation activities on the basis of the microsomal protein level at the single substrate concentration (100 μM) of variant CYP2D6s were less than 6% for CYP2D6.10 and 1% for CYP2D6.36 of those of CYP2D6.1. Kinetic analysis for mexiletine hydroxylation revealed that the affinity toward mexiletine of CYP2D6.10 and CYP2D6.36 was reduced by amino acid substitutions. The V max and V max / K m values of CYP2D6.10 on the basis of the microsomal protein level were reduced to less than 10% of those of CYP2D6.1, whereas the values on the basis of functional CYP2D6 level were comparable to those of CYP2D6.1. Although it was impossible to estimate the kinetic parameters for the mexiletine hydroxylation of CYP2D6.36, the metabolic ability toward mexiletine was considered to be poorer not only than that of CYP2D6.1 but also than that of CYP2D6.10. The same tendency was also observed in kinetic analysis for bufuralol 1″-hydroxylation as a representative CYP2D6 probe. These findings suggest that CYP2D6*36 has a more drastic impact on mexiletine metabolism than CYP2D6*10 .

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125CG, Pro42Arg, CYP1A2*15; 1130GA, Arg377Gln, *16; and 1367GA, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.

For this study we enrolled 1,615 males who were admitted to our hospital for a general health check-up. Plasma glucose (PG) and insulin were measured during 75 g OGTT, and abdominal obesity was assessed by ultrasonography in all subjects. We divided them into several groups: normal glucose tolerance (NGT), high-normal glucose tolerance (h-NGT) who showed >10.0 nmol/l at 1 hr PG among those with NGT, impaired fasting glucose (IFG), impaired glucose tolerance (IGT), IFG + IGT, and DM, according to the results of 75 g OGTT. The aim of the present study was to clarify the clinical characteristics of pre-diabetic disorders relating to metabolic syndrome by comparing various parameters including body mass index (BMI), blood levels of various lipids and abdominal wall fat index (AFI) calculated from the thickness of preperitoneal (Pmax) and subcutaneous (Smin) fat layer in the abdomen estimated by ultrasonography with insulin sensitivity determined by homeostatic model assessment (HOMA-IR) in each type of abnormal glucose regulation as classified by PG changes in 75 g OGTT. We also investigated the relationship between insulin secretion capability and insulin sensitivity to delineate the characteristics of each type of abnormal glucose regulation, and compared the area under the insulin curve (AUCins) and the time axis, and the ability of early insulin secretion by glucose loading (insulinogenic index: I.I.) in each type of abnormal glucose regulation. There was a significant positive correlation between HOMA-IR and Smin or Pmax, suggesting that Smin and Pmax may reflect insulin sensitivity. Abdominal obesity, which was diagnosed from the data of AFI, was present in the h-NGT and IFG + IGT groups, suggesting that those groups belong to the clinical entity of metabolic syndrome. HOMA-IR was higher in IFG than in IGT, although I.I. was reduced and AUCins was increased in IFG as well as in IGT. h-NGT demonstrated a slightly lower I.I. and higher AUCins, compared with IGT. IFG demonstrated much stronger insulin resistance than IGT, although I.I. was reduced and AUCins was increased in IFG and IGT. Thus, it is suggested that insulin sensitivity may partly account for the difference in pathogenesis between IFG and IGT; and that h-NGT, which showed abdominal obesity assessed as AFI by ultrasonography, should be recognized as a disease state of metabolic syndrome with impaired glucose regulation.

In order to identify genetic polymorphisms and haplotype frequencies of CYP1A2 in a Japanese population, the enhancer and promoter regions, all the exons with their surrounding introns, and intron 1 were sequenced from genomic DNA from 250 Japanese subjects. Thirty-three polymorphisms were found, including 13 novel ones: 2 in the enhancer region, 5 in the exons, and 6 in the introns. The most common single nucleotide polymorphism (SNP) was -163C>A (CYP1A2*1F allele) with a 0.628 frequency. In addition to six previously reported non-synonymous SNPs, three novel ones, 125C>G (P42R, CYP1A2*15 allele, MPJ6_1A2032), 1130G>A (R377Q, *16 allele, MPJ6_1A2033), and 1367G>A (R456H, *8 allele, MPJ6_1A2019), were found with frequencies of 0.002, 0.002, and 0.004, respectively. No polymorphism was found in the known nuclear transcriptional factor-binding sites in the enhancer region. Based on linkage disequilibrium analysis, the CYP1A2 gene was analyzed as one haplotype block. Using the 33 detected polymorphisms, 14 haplotypes were unambiguously identified, and 17 haplotypes were inferred by aid of an expectation-maximization-based program. Among them, the second major haplotype CYP1A2*1L is composed of -3860G>A (*1C allele), -2467delT (*1D allele), and -163C>A (*1F allele). Network analysis suggested that relatively rare haplotypes were derived from three major haplotypes, *1A, *1M, and *1N in most cases. Our findings provide fundamental and useful information for genotyping CYP1A2 in the Japanese, and probably Asian populations.

The development of functional mitral regurgitation (MR) in dilated cardiomyopathy (DCM) has been attributed to altered left ventricular (LV) geometry and annular dilatation. We propose the hypothesis that intraventricular dyssynchrony may play a role in the development of MR in DCM.Tissue Doppler echocardiography was performed in 32 DCM patients to assess the time from the onset of the QRS complex to the peak systolic myocardial strain (Ts) at 2 segments adjacent to the anterolateral and posteromedial papillary muscles from a short axis view. The time difference corrected by the RR interval (DeltaTs/ radicalRR) was used to evaluate dyssynchrony of these segments. There was no difference in the QRS duration (103 +/- 29 ms versus 95 +/- 22 ms, P = .38) or the presence of left bundle branch block (39% versus 14 %, P = .25) between 18 patients with MR and 14 patients without MR. However, DeltaTs/ radicalRR was significantly increased in the patients with MR, compared with those without MR (104 +/- 67 ms versus 5 +/- 16 ms, P.0001). Stepwise multiple regression analysis showed that DeltaTs/ radicalRR was independent contributing factor of MR.Dyssynchrony of myocardial segments adjacent to the papillary muscles may disturb synchronized closure of the mitral leaflets and cause MR in DCM.

症例は36歳,男性.健診の腹部エコーで両側副腎部に腫瘍を指摘された.尿中カテコラミン高値であり,腹部CT上両側副腎部に腫瘤を認め,またMIBGシンチ上同部に集積を認めたことから褐色細胞腫と診断した.生涯の補充療法の負担と副腎クリーゼのリスクを考慮して右副腎全摘,左側腫瘍核出術を施行した.本手術施行例は稀少で報告例も少ない.また家系内に両側褐色細胞腫が多発する興味深い症例であるので報告した.

Five novel single nucleotide polymorphisms (SNPs) were found in exon 3 and introns 1, 3, 7, and 8 in cytochrome P450 (CYP) 2C8 gene from 54 Japanese individuals, who were administered the anti-arrhythmic drug amiodarone. The detected SNPs were as follows: 1) SNP, MPJ6_2C8014; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5'-ATTCAGAAATATC/TGAATCTATGTGT-3' 2) SNP, MPJ6_2C8015; GENE NAME, CYP2C8; ACCESSION NUMBER, NM_000770 and NT_008769; LENGTH, 25 bases; 5'-GGAGGAGTTGAGA/-AAAACCAAGGGT-3'. 3) SNP, MPJ6_2C8016; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5'-ATTTGTAAGATAT/-TGTTTAAAATTT-3' 4) SNP, MPJ6_2C8017; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5'-TTGGTTCCAACCC/TTCTAACAACACA-3' 5) SNP, MPJ6_2C8018; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5'-GATAGCAAATATA/GTCTCTTTTTGTA-3' Among these SNPs, MPJ6_2C8015 was expected to cause a frame-shift due to the deletion of adenine 471, resulting in amino acid alterations from codon 159 and an early stop codon at residue 177. Therefore, the variant enzyme is most likely to be inactive since it lacks 64% of the protein structure, including the heme-binding site and 5 out of 6 substrate recognition sites.

Three non-synonymous single nucleotide polymorphisms (SNPs) in the CYP3A4 gene were found in 34 cell lines derived from Japanese individuals. These three SNPs (T185S, L293P, and T363M)(1) have been previously reported, but little is known about the effect that these polymorphisms, especially T185S, have on catalytic activity. We measured testosterone hydroxylation in wild-type CYP3A4 and these three variants using a mammalian expression system. Testosterone 6beta-, 2beta-, and 15beta-hydroxylations by the variant CYP3A4 forms T363M (40%) and T185S (60%) were reduced as compared with the wild-type in transient expression assays. L293P was similar to the wild-type in testosterone 6beta- and 2beta-hydroxylase activities. Western blot analysis confirmed lower amounts of CYP3A4 protein in the T363M and T185S variants than in the wild-type. Interestingly, Northern blot analysis showed no significant difference among mRNA levels between the wild-type and variants. These results suggest that the T363M and T185S substitutions in CYP3A4 affect either protein expression or stability. These established cell lines provided useful CYP3A4 SNP information in the Japanese.

We determined the CYP2D6 alleles in cell lines derived from 81 Japanese individuals with the CYP450 probe arrays using an Affymetrix GeneChip apparatus. Sequencing of the CYP2D6 exons from these same cell lines was performed to determine the accuracy of the allele calls by the Affymetrix probe array. Comparison of the results showed differences in the data from three cell lines for the CYP2D6(*)10 alleles between these two methods. These results indicated that the CYP450 probe array must be utilized cautiously for typing CYP2D6(*)10 alleles, which are frequently observed in the Japanese population.

Cytochrome P450 (CYP) 2D6 is an important drug-metabolizing enzyme, and its gene is known to be highly polymorphic. Here, we report five novel nonsynonymous single nucleotide polymorphisms (SNPs), and 65 other sequence variations detected from the gene coding for cytochrome P450 (CYP) 2D6 in 254 Japanese subjects. Two of the novel nonsynonymous SNPs were associated with the *10 key SNP, C100T. Among the 65 variations, 23 were novel, including 12 SNPs in 5'-flanking, 1 in 5'-untranslated, and 10 in intronic regions. The nonsynonymous SNPs in the CYP2D6 gene were as follows: 73 C>T (Arg25Trp, exon 1), 972 C>T (Ala90Val, exon 2), 1611 T>A (Phe120Ile, exon 3), 1720 A>C (Glu156Ala, exon 3), 3172 A>C (Glu334Ala, exon 7). The SNPs, 73C>T, 972 C>T, 1611 T>A, 1720 A>C and 3172 A>C were linked with *10, *1, *10, *1 and *2, respectively.

RA175 is a new member of the immunoglobulin superfamily with trans interaction activity, and it plays a role as a tumor suppressor in lung carcinoma (TSLC1) and as a cell adhesion molecule promoting the formation of functional synapses (SynCAM). Little is known about the biological function of RA175/TSLC1/SynCAM neural network formation during neurogenesis. We examined the distribution and colocalization of the RA175/TSLC1/SynCAM protein with other members of the immunoglobulin superfamily such as NCAM, L1, and TAG-1 in the mouse developing nervous system. Consistent with the expression of RA175/TSLC1/SynCAM mRNA, the protein was localized in the brain neuroepithelium at embryonic day (E) 9.5, neural crest at E10.5, motor neurons at E10.5, and olfactory epithelium at E16.5. In contrast with its mRNA, the protein was intensely detected on the fasciculated axons in the floor plates, ventral root, and dorsal funiculus in the E10.5-11.5 spinal cord and colocalized with NCAM and L1 on the ventral root and dorsal funiculus and partly colocalized with TAG-1 on the commissural axons and dorsal funiculus. In the E13.5-15.5 brain, RA175/TSLC1/SynCAM colocalized with NCAM and L1 on the developing thalamocortical fibers from the internal capsule (IC) and partly colocalized with TAG-1 on the cortical efferent axons in the intermediate zone (IZ). RA175/TSLC1/SynCAM was localized on the axons of some of the cortical neurons cultured in vitro. Thus, in addition to cell adhesion activity in the neuroepithelium and the synapses, RA175/TSLC1/SynCAM may be involved in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons.

Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the Vmax of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower Km value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

RA175, a new immunoglobulin superfamily member, is preferentially expressed during differentiation of P19 embryonic carcinoma (EC) cells induced by retinoic acid. In the present study, we isolated mouse RA175 cDNA in its entirety and showed that RA175 is the mouse ortholog of TSLC1, a tumor suppressor gene in human lung cancer. RA175/TSLC1 was localized in the adherent region of human lung squamous carcinoma cells and in the differentiated P19 EC cells. RA175/TSLC1 showed homophilic trans-interaction activity in a Ca(2+)-independent manner. RA175/TSLC1 was preferentially expressed in the polarized cells lining the lumen of developing mouse lung epithelium. This suggests that RA175/TSLC1 is a cell adhesion molecule that is acting as a tumor suppressor gene in the metastasis of lung tumors. RA175/TSLC1 may be necessary for cells to remain tightly associated in the epithelium, thereby suppressing metastasis.

Amiodarone is a potent Class III antiarrhythmic drug. The N-deethylation of amiodarone to desethylamiodarone is known to be catalyzed by cytochrome P450 (CYP) 2C8. In the present study, amiodarone N-deethylation by the CYP2C8s, CYP2C8*1 (wild-type), CYP2C8*3, and CYP2C8 P404A (Pro404Ala substitution in exon 8), was investigated by their transient expression in Hep G2 cells. The expression levels of CYP2C8*1 and CYP2C8*3 were similar, whereas the level of CYP2C8 P404A was 55.6% of that of CYP2C8*1. The kinetic parameters of amiodarone N-deethylation were obtained by means of Lineweaver-Burk analysis. The intrinsic clearance (Vmax/Km, per mg of microsomal protein) of amiodarone by CYP2C8 P404A but not CYP2C8*3 was significantly (48.7%) less than that of CYP2C8*1. These results suggest that CYP2C8 P404A but not CYP2C8*3 is less effective in the N-deethylation of amiodarone.

A selective and sensitive assay for amiodarone N-deethylation activity in human liver microsomes by high-performance liquid chromatography (HPLC) with UV detection is reported. The extraction of desethylamiodarone from incubation samples was performed by means of an original solid-phase extraction (SPE) procedure using a polymeric reversed-phase sorbent (Oasis HLB). The method was validated for the determination of desethylamiodarone with respect to specificity, linearity, precision, accuracy, recovery, limit of quantitation and stability. Amiodarone N-deethylation activity from low to high substrate concentrations using human liver microsomes was precisely determined without a concentration step. This method is applicable to the study in vitro of the metabolism of amiodarone.

We found nucleotide variability in the 5'-upstream region and exonic sequences of a gene-encoding canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) by polymerase chain reaction-based sequencing using genomic DNA from 72 established cell lines derived from 72 Japanese individuals. Four single nucleotide polymorphisms (SNPs) were found in the 5'-untranslational region and 21 in the exonic regions. Of them, 14 were nonsynonymous SNPs. One deletion of seven consecutive adenines resulting in a frameshift variant was also found. Four SNPs, c-24t, g1249a (V417I), c2366t (S789F), and c3972t (I1324I), were the same as those recently reported. A strong association was found between c-24t (5'-untranslated region) and c3972t (exon 28), with the promoter activity of the former worth being compared.

We found nucleotide variability in the 5′-upstream region and exonic sequences of a gene-encoding canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) by polymerase chain reaction-based sequencing using genomic DNA from 72 established cell lines derived from 72 Japanese individuals. Four single nucleotide polymorphisms (SNPs) were found in the 5′-untranslational region and 21 in the exonic regions. Of them, 14 were nonsynonymous SNPs. One deletion of seven consecutive adenines resulting in a frameshift variant was also found. Four SNPs, c-24t, g1249a (V417I), c2366t (S789F), and c3972t (I1324I), were the same as those recently reported. A strong association was found between c-24t (5′-untranslated region) and c3972t (exon 28), with the promoter activity of the former worth being compared.